Methods of treating cognitive impairment

ABSTRACT

The subject invention concerns materials and methods for treating a person or animal having cognitive impairment. In one embodiment, the method comprises administering an effective amount of one or more inflammatory mediator(s), for example, fms-related tyrosine kinase 3 (Flt3) ligand, interleukin-6 (IL-6), macrophage migration inhibitory factor (MIF), interleukin-1 (IL-1), interleukin-3 (IL-3), erythropoietin (EPO), vascular endothelial growth factor A (VEGF-A), hypoxia-inducible transcription factor (HIF-1alpha), insulin like growth factor-1 (IGF-1), tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), Stem Cell Factor (SCF), Darbepoetin (ARANESP), and metalloproteinases, to an animal or person in need of treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/057,387, filed Feb. 3, 2011, which is the National Stage ofInternational Application Number PCT/US2009/052742, filed Aug. 4, 2009,which claims the benefit of U.S. Provisional Application Ser. No.61/086,351, filed Aug. 5, 2008, each of which is hereby incorporated byreference herein in its entirety, including any figures, tables, nucleicacid sequences, amino acid sequences, and drawings.

BACKGROUND OF THE INVENTION

Memory loss has long been recognized as a common accompaniment of aging.The inabilities to recall the name of a recent acquaintance or thecontents of a short shopping list are familiar experiences for everyone,and this experience seems to become more common as we age.

Over the last few decades, the medical community has changed its view ofmemory loss in the elderly. These problems were viewed in the past asinevitable accompaniments of aging, often referred to as “senility” or“senior moments.” More recently, physicians have shifted their view ofmemory loss, such that memory impairment of a certain degree is nowconsidered pathological, and thus indicative of a disease processaffecting the brain. The threshold most physicians use to make thisjudgment is that memory loss has progressed to such an extent thatnormal independent function is impossible; for instance, if one can nolonger successfully manage one's own finances or provide for one's ownbasic needs. This degree of cognitive impairment has come to be referredto as dementia.

However, many older individuals may complain of memory problems, butstill manage to independently accomplish all their customary tasks.Usually, their ability to function well is based on compensation forthese difficulties, such as increased reliance on a calendar or onreminder notes, lists, etc. In some cases, these memory difficulties area sign that worsening memory loss is on the horizon.

The syndrome of subjective memory problems has come to be commonly knownas “Mild Cognitive Impairment” (MCI), although other terms have beenused, including “Cognitive Impairment, Not Dementia” (CIND). The patientwith MCI complains of difficulty with memory. Typically, the complaintsinclude trouble remembering the names of people they met recently,trouble remembering the flow of a conversation, and an increasedtendency to misplace things, or similar problems. In many cases, theindividual will be quite aware of these difficulties and will compensatewith increased reliance on notes and calendars. Most importantly, thediagnosis of MCI relies on the fact that the individual is able toperform all their usual activities successfully, without more assistancefrom others than they previously needed.

Several studies have examined the cognitive performance of patients withMCI. These have demonstrated that, in general, these patients performrelatively poorly on formal tests of memory, even when compared withother individuals in their age group. They also show mild difficultiesin other areas of thinking, such as naming objects or people (coming upwith the names of things) and complex planning tasks. These problems aresimilar, but less severe, than the neuropsychological findingsassociated with Alzheimer's disease. Careful questioning has alsorevealed that, in some cases, mild difficulties with daily activities,such as performing hobbies, are evident.

Several studies have demonstrated that memory complaints in the elderlyare associated with a higher-than-normal risk of developing dementia inthe future. Most commonly, the type of dementia that patients with MCIare at risk to develop is Alzheimer's disease, though other dementias,such as Vascular Dementia or Frontotemporal Dementia may occur as well.However, it is also clear that some patients with these complaints neverdevelop dementia.

Certain features are associated with a higher likelihood of progression.These include confirmation of memory difficulties by a knowledgeableinformant (such as a spouse, child, or close friend), poor performanceon objective memory testing, and any changes in the ability to performdaily tasks, such as hobbies or finances, handling emergencies, orattending to one's personal hygiene.

One factor that had to be controlled for in many of these studies wasdepression, as many patients with depression also complain about theirmemory. Several studies have suggested that certain measurements ofatrophy (shrinkage) or decreased metabolism on images of the brain (PETor MRI scans) increase the chances of developing dementia in the future.

Although these above factors increase the chances of going on to developdementia, it is not possible currently to predict with certainty whichpatients with MCI will or will not go on to develop dementia. Thus, manyof these measures, particularly the measurements from brain images, arestill considered to be useful only for research.

One neurological disorder that is most widely known for its progressiveloss of intellectual capacities is Alzheimer's disease (AD). Worldwide,about 20 million people suffer from Alzheimer's disease. AD isclinically characterized by the initial loss of memory, followed bydisorientation, impairment of judgment and reasoning, which is commonlyreferred to as cognitive impairment, and ultimately by full dementia. ADpatients finally lapse into a severely debilitated, immobile statebetween four and twelve years after onset of the disease.

The key pathological evidence for AD is the presence of extracellularamyloid plaques and intracellular tau tangles in the brain, which areassociated with neuronal degeneration (Ritchie and Lovestone (2002)).The extracellular amyloid plaques are believed to result from anincrease in the insoluble amyloid beta peptide 1-42 produced by themetabolism of amyloid-beta precursor protein (APP). Following β, γsecretion, these amyloid beta 1-42 peptides form amyloid fibrils morereadily than the amyloid beta 1-40 peptides, which are predominantlyproduced in healthy people. It appears that the amyloid beta peptide ison top of the neurotoxic cascade: experiments show that amyloid betafibrils, when injected into the brains of P301 L tau transgenic mice,enhance the formation of neurofibrillary tangles (Götz et al. (2001)).In fact, a variety of amyloid beta peptides have been identified asamyloid beta peptides 1-42, 1-40, 1-39, 1-38, 1-37, which can be foundin plaques and are often seen in cerebral spinal fluid.

The amyloid beta peptides are generated (or processed) from the membraneanchored APP, after cleavage by beta secretase and gamma secretase atposition 671 and 711 or 713, respectively. In addition, high activity ofbeta secretase results in a shift of the cleavage at position 1 toposition 11. Cleavage of amyloid-beta precursor protein by alphasecretase activity will generate Aβ 1-17 and gamma secretase activity at40 or 42 generates the non-pathological p3 peptide. Beta secretase wasidentified as the membrane anchored aspartyl protease BACE, while gammasecretase is a protein complex comprising presenilin 1 (PS1) orpresenilin 2 (PS2), nicastrin, Anterior Pharynx Defective 1 (APH1) andPresenilin Enhancer 2 (PEN2). Of these proteins, the presenilins arewidely thought to constitute the catalytic activity of the gammasecretase, while the other components play a role in the maturation andlocalization of the complex. The identity of the alpha secretase isstill illustrious, although some results point towards the proteasesADAM 10 and TACE, which could have redundant functions.

A small fraction of AD cases (mostly early onset AD) are caused byautosomal dominant mutations in the genes encoding presenilin 1 and 2(PS 1; PS2) and the amyloid-beta precursor protein (APP), and it hasbeen shown that mutations in APP, PS1 and PS2 alter the metabolism ofamyloid-beta precursor protein leading to such increased levels ofamyloid beta 1-42 produced in the brain. Although no mutations in PS1,PS2 and amyloid-beta precursor protein have been identified in lateonset AD patients, the pathological characteristics are highly similarto the early onset AD patients. These increased levels of amyloid betapeptide could originate progressively with age from disturbedamyloid-beta precursor protein processing (e.g. high cholesterol levelsenhance amyloid beta peptide production) or from decreased amyloid betapeptide catabolism. Therefore, it is generally accepted that AD in lateonset AD patients is also caused by aberrant increased amyloid peptidelevels in the brains. The level of these amyloid beta peptides, and moreparticularly amyloid-beta peptide 1-42, is increased in Alzheimerpatients compared to the levels of these peptides in healthy persons.

BRIEF SUMMARY OF THE INVENTION

The present invention provides materials and methods for treating aperson or animal having cognitive impairment wherein the methodcomprises administering an effective amount of an inflammatory mediatorthat is able to cross the blood brain barrier. In one embodiment, theinflammatory mediator provides for an increase in cognitive responses inthe brain and/or exerting a neural protective effect. Inflammatorymediators contemplated within the scope of the present inventioninclude, but are not limited to, fms-related tyrosine kinase 3 (Flt3)ligand, interleukin-6 (IL-6), macrophage migration inhibitory factor(MIF), interleukin-1 (IL-1), interleukin-3 (IL-3), erythropoietin (EPO),vascular endothelial growth factor A (VEGF-A), hypoxia-inducibletranscription factor (HIF-1alpha), insulin like growth factor-1 (IGF-1),tumor necrosis factor (TNF), granulocyte colony-stimulating factor(G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF),macrophage colony-stimulating factor (M-CSF), Stem Cell Factor (SCF),Darbepoetin (ARANESP), and metalloproteinases. In one embodiment, theinflammatory mediator is GM-CSF, or a functional fragment or variantthereof. In a specific embodiment, the cognitive impairment is caused byor results from Alzheimer's disease. In addition, it is alsocontemplated that compounds capable of inducing GM-CSF production in amammal, which subsequently exert an increase in cognitive responses inthe brain, can be used according to the subject invention. In oneembodiment, the inflammatory mediator is administered or delivered to anon-neural cell or tissue of the human or animal.

The subject invention also concerns methods for decreasing or inhibitingthe progression of cognitive impairment in a person or animal havingmemory problems associated with cognitive functions or relateddementias, comprising administering an effective amount of aninflammatory mediator as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show pictures of a typical radial arm water maze devicefor cognitive disease behavioral testing in mice. FIG. 1 shows thediagram of the maze as one would look down on the device. FIG. 2 showsthe device itself which is used for the behavioral testing of animals.

FIGS. 3 and 4 show GMCSF Interference testing—Block 1 (FIG. 3) and Block2 (FIG. 4).

FIGS. 5A and 5B show decreasing levels of insoluble amyloid beta levelsin the hippocampus of mice. FIG. 5C shows an increase in levels ofsoluble amyloid beta levels in hippocampus of mice with increasingplasma concentrations of GM-CSF.

FIGS. 6A and 6B: FIG. 6A shows intrahippocampal injection of GM-CSF(left hemisphere) and aCSF (right hemisphere). Representative coronaltissue cryosectioned at 14 μm and stained with MabTech α-Aβ/Alexa 546.Image is a montage of about 145 pictures taken at 10×. White spotsindicate amyloid plaque immunolabelling (see FIGS. 13A-13D forrepresentative montaged sections of all 4 mice). FIG. 6B: overall plaquereductions seen in all 4 plaque parameters measured, computated from 5quantified sections per mouse (n=4 mice). Error bars are ±SEM.Statistical significance obtained by paired Students t-test with Pvalues (Area: P<1.11E-07; Perimeter: P<1.41E-06; Feret Diameter:P<2.36E-09; Integrated Density: P<1.11E-07).

FIGS. 7A-7F show behavioral analysis following daily subcutaneous GM-CSFinjections. FIGS. 7A-7D show standard Radial Arm Water Maze errors.Shows substantial impairment in Tg control mice (n=8) on working memorytrials T4 and T5 compared to NT control mice (n=8) in individual blocksof testing (FIGS. 7A and 7B), and over all 4 days of testing (FIGS. 7Cand 7D). GM-CSF-treated Tg mice (n=7) performed as well as or betterthan NT control mice on working memory trials T4 and T5 duringindividual blocks and over all. GM-CSF-treated NT mice (n=9) performedsimilarly to or slightly better than NT controls (Note significantlybetter performance of NT+GMCSF group vs. NT group for T4 of Block 1,FIG. 7A), although this effect was not significant overall. Statisticalsignificance determined by one-way ANOVA (**P<0.05 or highersignificance versus all other groups; ¶ P<0.05 or higher significanceversus Tg+GM-CSF and NT+GM-CSF). FIG. 7E shows cognitive InterferenceTask Overall (4 Days) Shows Tg control mice are impaired compared to NTmice on all four cognitive measures assessed. GM-CSF-treated Tg miceexhibited significantly better a-trial recall (A1-A3) and delayed recall(A5) compared to Tg controls and performed similarly to NT mice in allfour cognitive measures. GM-CSF treatment of NT mice did not result insignificantly better performance compared to NT controls, althoughtrends for a beneficial GM-CSF effect in NT mice were evident overall.Statistical significance was determined by one-way ANOVA (*Tgsignificantly different from NT+GM-CSF, **Tg significantly differentfrom all other groups). FIG. 7F shows cognitive Interference Task.Proactive Interference testing (First 2 days). GM-CSF-treated Tg miceperformed significantly better than Tg controls and equally to NT andGM-CSF-treated NT mice. Statistical significance determined by one-wayANOVA (**P<0.05 or higher significance versus all other groups).

FIGS. 8A-8E show amyloid deposition in subcutaneous GM-CSF-injectedmice. FIGS. 8A-8D are photomicrographs of coronal 5 μm paraffin-embeddedsections immunolabelled with anti-Aβ antibody (clone 4G8). Pictures arerepresentative of amyloid load closest to the mean of the GM-CSF- orsaline-treated Tg groups. Scale bar=50 μm. FIG. 8E shows the percent ofamyloid burden from the average of 5 sections per mouse ofGM-CSF-treated (n=5) versus saline-treated (n=6). Statisticalsignificance was determined by two-tailed homoscedastic Student'st-test: Entorhinal cortex (*P<0.026), and Hippocampus (P=0.12).

FIGS. 9A-9E shows synaptophysin immunostaining in subcutaneousGM-CSF-injected mice. FIGS. 9A-9D are photomicrographs of coronal 5 μmparaffin-embedded sections immunolabelled with anti-synaptophysinantibody. Pictures are representative of synaptophysin immunolabellingclosest to the mean of the GM-CSF- or saline-treated groups. Scalebar=50 μm. FIG. 9E shows the percent of synaptophysin immunoreactivityfrom the average of 5 sections per mouse of GM-CSF-treated (n=5) versussaline-treated (n=6). Albeit numerically small, differences between thetwo groups were statistically significant by two-tailed homoscedasticStudent's t-test: CA1(P<0.0013), CA3(P<0.0023).

FIG. 10 shows significant variation of amyloid plaque load between mice.PS/APP mice of similar age (numbered sequentially according to date ofbirth) after bilateral intracerebroventricular infusions of M-CSF. Mousenumbers 148, 160, 171, and 211 received M-CSF and mice 164, 170, 176,and 177 received aCSF. Infusions lasted 2 weeks. Cryosectioned at 14 μmand stained with 6E10/Alexa 488 and Hoechst. Bright green spots indicateamyloid plaques. Pictures taken at 5×.

FIGS. 11A-11C show intrahippocampal injection of M-CSF (left hemisphere)and aCSF (right hemisphere). In FIG. 11A, the image is a montage of ˜14510× pictures and is representative of the effects seen from anteriorhippocampus to posterior in all 4 M-CSF-injected mice. TheM-CSF-injected hemispheres show no difference of amyloid depositionbetween hemispheres. FIG. 11B: This photo shows enlargement of theM-CSF-injected left hemisphere, as seen following saline perfusion. Notethe small bump at the site of injection. FIG. 11C is an image showingcyst or tumor-like growth formed in the needle track at the site ofM-CSF injection. Cryosectioned at 14 μm and stained with 6E10/Alexa 488and Hoechst. Bright green staining is non-specific and not indicative ofamyloid plaque. Picture taken at 20×.

FIGS. 12A and 12B show intrahippocampal injection of G-CSF (lefthemisphere) and aCSF (right hemisphere). Amyloid plaques indicated aswhite spots. Although the brain was sectioned at a slight angle, visualobservation of amyloid plaques show a general reduction throughout theleft G-CSF-injected hemisphere. Cryosectioned at 14 μm and stained with6E10/IR800. Scanned on the Licor Odyssey and enlarged for visualization.Sections numbered 1 through 6 and correspond with anterior to posterior.

FIGS. 13A-13D show intrahippocampal injection of GM-CSF (lefthemisphere) and aCSF (right hemisphere). Representative sections of eachmouse proximal to injection site. Tissue sections stained with MabTechα-Aβ/Alexa 488. White spots indicate amyloid plaque immunolabelling.Images are montages of about 145 pictures taken at 10×. FIGS. 13A-13Care from 14 μm frozen sections, and FIG. 13D is from a 5 μmparaffin-embedded section.

FIGS. 14A, 14A-1, 14B, 14B-1, 14C, 14C-1, 14D, 14D-1, 14E, 14E-1, 14Fand 14F-1 show quantification of reduced amyloid deposition inGM-CSF-injected left hemispheres versus aCSF—injected contralateralright hemispheres. There were 5 sections per mouse quantified. Eachmontaged section contained over 140 10× pictures and of these, 15-25pictures per hemisphere were selected to quantify. All pictures persection were taken at the same exposure on a Zeiss Imager.Z1fluorescence microscope with a Zeiss Axiocam Mrm camera (Oberkochen,Germany) using Axiovision 4.7 software. Each figure shows total oraverage values from the 5 sections/mouse with significance perindividual mouse and overall. Error bars are ±SEM: (FIGS. 14A, 14A-1,14B, and 14B-1) plaque areas (FIGS. 14C, 14C-1, 14D, 14D-1) perimetervalues (FIGS. 14E and 14E-1) average feret diameters (FIGS. 14F and14F-1) average integrated densities.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for treating a person or animalhaving cognitive impairment wherein the method comprises administeringto the person or animal an effective amount of one or more inflammatorymediators that are able to cross the blood brain barrier, or apolynucleotide(s) encoding the inflammatory mediator(s) (if theinflammatory mediator is a polypeptide), or a composition comprising theinflammatory mediator(s) or polynucleotide(s). The cognitive impairmentcan be a progressive cognitive impairment. In a specific embodiment, thecognitive impairment is caused by or results from Alzheimer's disease.In one embodiment, the cognitive impairment is caused by or results fromstroke, Down's syndrome, dementia pugilistica, traumatic brain injury,AIDS-associated dementia, Lewy body disease, or Pick's disease. In oneembodiment, the inflammatory mediator provides for an increase incognitive responses in the brain and/or a neural protective effect. Inone embodiment, the inflammatory mediator, or polynucleotide encodingthe inflammatory mediator, is of human origin or sequence.

Inflammatory mediators contemplated within the scope of the presentinvention include, but are not limited to, fms-related tyrosine kinase 3(Flt3) ligand, interleukin-6 (IL-6), macrophage migration inhibitoryfactor (MIF), interleukin-1 (IL-1), interleukin-3 (IL-3), erythropoietin(EPO), vascular endothelial growth factor A (VEGF-A), hypoxia-inducibletranscription factor (HIF-1alpha), insulin like growth factor-1 (IGF-1),tumor necrosis factor (TNF), granulocyte colony-stimulating factor(G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF),macrophage colony-stimulating factor (M-CSF), Stem Cell Factor (SCF),Darbepoetin (ARANESP), and metalloproteinases, or a functional fragmentor variant thereof that exhibits substantially the same biologicalactivity as the full-length or non-variant inflammatory mediator.Typically, to treat cognitive impairment in a human, the inflammatorymediator will have the sequence of the human protein or polynucleotideencoding it. The human sequences of the inflammatory mediators describedherein are known in the art (see, for example, GenBank Accession Nos.NM_(—)013520.3, NM_(—)000799.2, NM_(—)000759.2, M11220.1,NM_(—)181054.2, NG_(—)011713.1, NG_(—)008851.1, NM_(—)000588.3,NG_(—)011640.1, NM_(—)000757.4, NG_(—)012099.1, NM_(—)004530.4,NG_(—)011468.1, NG_(—)007462.1, and NG_(—)008732.1). In one embodiment,the inflammatory mediator is GM-CSF, or a functional fragment or variantthereof that exhibits GM-CSF biological activity. In a specificembodiment, the GM-CSF is human GM-CSF. In one embodiment, GM-CSF andDarbepoetin and/or EPO are administered in a method of the invention fortreating cognitive impairment. In another embodiment, G-CSF andDarbepoetin and/or EPO are administered. In addition, it is alsocontemplated that compounds capable of inducing production in a mammalof an inflammatory mediator of the invention, such as GM-CSF, whichsubsequently exerts an increase in cognitive responses in the brain, canbe used according to the subject invention. Examples of such compoundsinclude, but are not limited to, EPO and HIF-1α (Fisher (2003)). In oneembodiment, the inflammatory mediator is administered or delivered to anon-neural tissue of the human or animal.

In one embodiment, an inflammatory mediator of the invention, or apolynucleotide encoding the inflammatory mediator, or a compositioncontaining the inflammatory mediator or polynucleotide, is delivered atan effective dose subcutaneously or through infusion, intracranially,orally, parentally, intranasally, via inhalation, intrathecally,intramuscularly, sublingually, or by any other known and acceptablemethods of drug delivery to a person or animal in need of such therapy.

The subject invention also concerns methods for decreasing or inhibitingthe progression of cognitive impairment in a person or animal havingmemory problems associated with cognitive functions or relateddementias. In one embodiment, a method of the invention comprisesadministering an effective amount of an inflammatory mediator of theinvention, or a polynucleotide encoding the inflammatory mediator, or acomposition containing the inflammatory mediator or polynucleotide, tothe person or animal.

In vivo application of the inflammatory mediators of the invention,polynucleotides encoding the inflammatory mediators, and/or compositionscontaining them, can be accomplished by any suitable method andtechnique presently or prospectively known to those skilled in the art.The subject compounds can be formulated in a physiologically- orpharmaceutically-acceptable form and administered by any suitable routeknown in the art including, for example, oral, nasal, rectal, andparenteral routes of administration. As used herein, the term parenteralincludes subcutaneous, intradermal, intravenous, intramuscular,intraperitoneal, and intrasternal administration, such as by injection.Administration of the subject inflammatory mediators of the inventioncan be a single administration, or at continuous or distinct intervalsas can be readily determined by a person skilled in the art.

The inflammatory mediators of the subject invention, polynucleotidesencoding the inflammatory mediators, and compositions comprising them,can also be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time. The inflammatory mediators of the invention canalso be administered in their salt derivative forms or crystallineforms.

Inflammatory mediators of the subject invention and polynucleotidesencoding the inflammatory mediators can be formulated according to knownmethods for preparing physiologically acceptable and/or pharmaceuticallyacceptable compositions. Formulations are described in detail in anumber of sources which are well known and readily available to thoseskilled in the art. For example, Remington's Pharmaceutical Science byE. W. Martin describes formulations which can be used in connection withthe subject invention. In general, the compositions of the subjectinvention will be formulated such that an effective amount of thecompound is combined with a suitable carrier in order to facilitateeffective administration of the composition. The compositions used inthe present methods can also be in a variety of forms. These include,for example, solid, semi-solid, and liquid dosage forms, such astablets, pills, powders, liquid solutions or suspension, suppositories,injectable and infusible solutions, and sprays. The preferred formdepends on the intended mode of administration and therapeuticapplication. The compositions also preferably include conventionalphysiologically-acceptable carriers and diluents which are known tothose skilled in the art. Examples of carriers or diluents for use withthe subject compounds include ethanol, dimethyl sulfoxide, glycerol,alumina, starch, saline, and equivalent carriers and diluents. Toprovide for the administration of such dosages for the desiredtherapeutic treatment, compositions of the invention may comprisebetween about 0.1% and 99%, and especially, 1 and 15% by weight of thetotal of one or more of the subject inflammatory mediator based on theweight of the total composition including carrier or diluent.

Inflammatory mediators of the invention, polynucleotides encoding theinflammatory mediators, and compositions comprising them, can bedelivered to a cell either through direct contact with the cell or via acarrier means. Carrier means for delivering inflammatory mediators andcompositions to cells are known in the art and include, for example,encapsulating the composition in a liposome moiety. Another means fordelivery of inflammatory mediators and compositions of the invention toa cell comprises attaching the inflammatory mediators to a protein ornucleic acid that is targeted for delivery to the target cell. U.S. Pat.No. 6,960,648 and Published U.S. Patent Application Nos. 20030032594 and20020120100 disclose amino acid sequences that can be coupled to anothercomposition and that allows the composition to be translocated acrossbiological membranes. Published U.S. Patent Application No. 20020035243also describes compositions for transporting biological moieties acrosscell membranes for intracellular delivery. Inflammatory mediators canalso be incorporated into polymers, examples of which include poly (D-Llactide-co-glycolide) polymer for intracranial delivery;poly[bis(p-carboxyphenoxy) propane:sebacic acid] in a 20:80 molar ratio(as used in GLIADEL); chondroitin; chitin; and chitosan.

While inflammatory mediators of the invention and polynucleotidesencoding the inflammatory mediators can be administered by themselves,these inflammatory mediators can also be administered as part of apharmaceutical composition. The subject invention thus further providescompositions comprising one or more inflammatory mediators inassociation with at least one pharmaceutically acceptable carrier. Thepharmaceutical composition can be adapted for various routes ofadministration, such as enteral, parenteral, intravenous, intramuscular,topical, subcutaneous, and so forth. Administration can be continuous orat distinct intervals, as can be determined by a person of ordinaryskill in the art.

Other formulations of inflammatory mediators and polynucleotidesencoding them suitable for administration include, for example, aqueoussterile injection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

Inflammatory mediators of the invention, polynucleotides encoding theinflammatory mediators, and compositions thereof, may be locallyadministered at one or more anatomical sites, optionally in combinationwith a pharmaceutically acceptable carrier such as an inert diluent.Inflammatory mediators of the invention, and compositions thereof, maybe systemically administered, such as intravenously or orally,optionally in combination with a pharmaceutically acceptable carriersuch as an inert diluent, or an assimilable edible carrier for oraldelivery. They may be enclosed in hard or soft shell gelatin capsules,may be compressed into tablets, or may be incorporated directly with thefood of the patient's diet. For oral therapeutic administration, theinflammatory mediators may be combined with one or more excipients andused in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and thelike.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac, or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

Inflammatory mediators, and polynucleotides encoding the inflammatorymediators, and compositions of the invention, including pharmaceuticallyacceptable salts or analogs thereof, can be administered intravenously,intramuscularly, or intraperitoneally by infusion or injection.Solutions of the active agent or its salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating aninflammatory mediator of the invention in the required amount in theappropriate solvent with various other ingredients enumerated above, asrequired, followed by filter sterilization. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze dryingtechniques, which yield a powder of the active ingredient plus anyadditional desired ingredient present in the previously sterile-filteredsolutions.

For topical administration, inflammatory mediators of the invention andpolynucleotides encoding the inflammatory mediators may be applied in asa liquid or solid. However, it will generally be desirable to administerthem topically to the skin as compositions, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.Inflammatory mediators can be applied in a formulation such as anointment, cream, lotion, solution, tincture, or the like. Drug deliverysystems for delivery of pharmacological substances to dermal sites canalso be used, such as that described in U.S. Pat. No. 5,167,649.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver an inflammatory mediator to the skin aredisclosed in U.S. Pat. No. 4,608,392; U.S. Pat. No. 4,992,478; U.S. Pat.No. 4,559,157; and U.S. Pat. No. 4,820,508.

Useful dosages of the inflammatory mediators, polynucleotides encodingthe inflammatory mediators, and pharmaceutical compositions of thepresent invention can be determined by comparing their in vitroactivity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art; for example, see U.S. Pat. No. 4,938,949.

The present invention also concerns pharmaceutical compositionscomprising an inflammatory mediator of the invention, or apolynucleotide encoding the inflammatory mediator, in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor oral, topical or parenteral administration, comprising an amount ofa compound constitute a preferred embodiment of the invention. The doseadministered to a patient, particularly a human, in the context of thepresent invention should be sufficient to achieve a therapeutic responsein the patient over a reasonable time frame, without lethal toxicity,and preferably causing no more than an acceptable level of side effectsor morbidity. One skilled in the art will recognize that dosage willdepend upon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

To provide for the administration of such dosages for the desiredtherapeutic treatment, in some embodiments, pharmaceutical compositionsof the invention can comprise between about 0.1% and 45%, andespecially, 1 and 15%, by weight of the total of one or more of thecompounds based on the weight of the total composition including carrieror diluents. Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.01 to about 20 mg/kg;intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about200 mg/kg, and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

The subject invention also concerns polynucleotide expression constructsthat comprise a polynucleotide of the present invention comprising anucleotide sequence encoding an inflammatory mediator of the presentinvention. In one embodiment, the polynucleotide encodes human GM-CSF,or a fragment or variant thereof that exhibits substantially the sameactivity as the full-length or non-variant GM-CSF.

As used herein, the term “expression construct” refers to a combinationof nucleic acid sequences that provides for transcription of an operablylinked nucleic acid sequence. As used herein, the term “operably linked”refers to a juxtaposition of the components described wherein thecomponents are in a relationship that permits them to function in theirintended manner. In general, operably linked components are incontiguous relation.

Expression constructs of the invention will also generally includeregulatory elements that are functional in the intended host cell inwhich the expression construct is to be expressed. Thus, a person ofordinary skill in the art can select regulatory elements for use in, forexample, bacterial host cells, yeast host cells, plant host cells,insect host cells, mammalian host cells, and human host cells.Regulatory elements include promoters, transcription terminationsequences, translation termination sequences, enhancers, andpolyadenylation elements.

An expression construct of the invention can comprise a promotersequence operably linked to a polynucleotide sequence encoding aninflammatory mediator of the invention. Promoters can be incorporatedinto a polynucleotide using standard techniques known in the art.Multiple copies of promoters or multiple promoters can be used in anexpression construct of the invention. In a preferred embodiment, apromoter can be positioned about the same distance from thetranscription start site as it is from the transcription start site inits natural genetic environment. Some variation in this distance ispermitted without substantial decrease in promoter activity. Atranscription start site is typically included in the expressionconstruct.

For expression in animal cells, an expression construct of the inventioncan comprise suitable promoters that can drive transcription of thepolynucleotide sequence. If the cells are mammalian cells, thenpromoters such as, for example, actin promoter, metallothioneinpromoter, NF-kappaB promoter, EGR promoter, SRE promoter, IL-2 promoter,NFAT promoter, osteocalcin promoter, SV40 early promoter and SV40 latepromoter, Lck promoter, BMP5 promoter, TRP-1 promoter, murine mammarytumor virus long terminal repeat promoter, STAT promoter, or animmunoglobulin promoter can be used in the expression construct. Thebaculovirus polyhedrin promoter can be used with an expression constructof the invention for expression in insect cells. Promoters suitable foruse with an expression construct of the invention in yeast cellsinclude, but are not limited to, 3-phosphoglycerate kinase promoter,glyceraldehyde-3-phosphate dehydrogenase promoter, metallothioneinpromoter, alcohol dehydrogenase-2 promoter, and hexokinase promoter.

For expression in prokaryotic systems, an expression construct of theinvention can comprise promoters such as, for example, alkalinephosphatase promoter, tryptophan (trp) promoter, lambda P_(L) promoter,β-lactamase promoter, lactose promoter, phoA promoter, T3 promoter, T7promoter, or tac promoter (de Boer et al., 1983).

If the expression construct is to be provided in a plant cell, plantviral promoters, such as, for example, the cauliflower mosaic virus(CaMV) 35S (including the enhanced CaMV 35S promoter (see, for exampleU.S. Pat. No. 5,106,739)) or 19S promoter can be used. Plant promoterssuch as prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA1′- or 2′-promoter of A. tumafaciens, polygalacturonase promoter,chalcone synthase A (CHS-A) promoter from petunia, tobacco PR-1αpromoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2promoter (Xu et al., 1993), maize WipI promoter, maize trpA genepromoter (U.S. Pat. No. 5,625,136), maize CDPK gene promoter, andRUBISCO SSU promoter (U.S. Pat. No. 5,034,322) can also be used.Seed-specific promoters such as the promoter from a β-phaseolin gene (ofkidney bean) or a glycinin gene (of soybean), and others, can also beused. Constitutive promoters (such as the CaMV, ubiquitin, actin, or NOSpromoter), tissue-specific promoters (such as the E8 promoter fromtomato), developmentally-regulated promoters, and inducible promoters(such as those promoters than can be induced by heat, light, hormones,or chemicals) are contemplated for use with the polynucleotides of theinvention.

Expression constructs of the invention may optionally contain atranscription termination sequence, a translation termination sequence,signal peptide sequence, and/or enhancer elements. Transcriptiontermination regions can typically be obtained from the 3′ untranslatedregion of a eukaryotic or viral gene sequence. Transcription terminationsequences can be positioned downstream of a coding sequence to providefor efficient termination. Signal peptides are a group of short aminoterminal sequences that encode information responsible for therelocation of an operably linked peptide to a wide range ofpost-translational cellular destinations, ranging from a specificorganelle compartment to sites of protein action and the extracellularenvironment. Targeting a polypeptide to an intended cellular and/orextracellular destination through the use of operably linked signalpeptide sequence is contemplated for use with the inflammatory mediatorsof the invention. Chemical enhancers are cis-acting elements thatincrease gene transcription and can also be included in the expressionconstruct. Chemical enhancer elements are known in the art, and include,but are not limited to, the CaMV 35S enhancer element, cytomegalovirus(CMV) early promoter enhancer element, and the SV40 enhancer element.DNA sequences which direct polyadenylation of the mRNA encoded by thestructural gene can also be included in the expression construct.

Unique restriction enzyme sites can be included at the 5′ and 3′ ends ofthe expression construct to allow for insertion into a polynucleotidevector. As used herein, the term “vector” refers to any genetic element,including for example, plasmids, cosmids, chromosomes, phage, virus, andthe like, which is capable of replication when associated with propercontrol elements and which can transfer polynucleotide sequences betweencells. Vectors contain a nucleotide sequence that permits the vector toreplicate in a selected host cell. A number of vectors are available forexpression and/or cloning, and include, but are not limited to, pBR322,pUC series, M13 series, and pBLUESCRIPT vectors (Stratagene, La Jolla,Calif.).

Polynucleotides, vectors, and expression constructs of the subjectinvention can be introduced into a cell by methods known in the art.Such methods include transfection, microinjection, electroporation,lipofection, cell fusion, calcium phosphate precipitation, and bybiolistic methods. In one embodiment, a polynucleotide or expressionconstruct of the invention can be introduced in vivo via a viral vectorsuch as adeno-associated virus (AAV), herpes simplex virus (HSV),papillomavirus, adenovirus, and Epstein-Barr virus (EBV). Attenuated ordefective forms of viral vectors that can be used with the subjectinvention are known in the art. Typically, defective virus is notcapable of infection after the virus is introduced into a cell.Polynucleotides, vectors, and expression constructs of the invention canalso be introduced in vivo via lipofection (DNA transfection vialiposomes prepared from synthetic cationic lipids) (Felgner et al.,1987). Synthetic cationic lipids (LIPOFECTIN, Invitrogen Corp., LaJolla, Calif.) can be used to prepare liposomes to encapsulate apolynucleotide, vector, or expression construct of the invention. Apolynucleotide, vector, or expression construct of the invention canalso be introduced in vivo as naked DNA using methods known in the art,such as transfection, microinjection, electroporation, calcium phosphateprecipitation, and by biolistic methods.

Polynucleotides and polypeptides of the subject invention can also bedefined in terms of more particular identity and/or similarity rangeswith those exemplified herein. The sequence identity will typically begreater than 60%, preferably greater than 75%, more preferably greaterthan 80%, even more preferably greater than 90%, and can be greater than95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequenceexemplified herein. Unless otherwise specified, as used herein percentsequence identity and/or similarity of two sequences can be determinedusing the algorithm of Karlin and Altschul (1990), modified as in Karlinand Altschul (1993). Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (1990). BLAST searches can beperformed with the NBLAST program, score=100, wordlength=12, to obtainsequences with the desired percent sequence identity. To obtain gappedalignments for comparison purposes, Gapped BLAST can be used asdescribed in Altschul et al. (1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) can be used. See NCBI/NIH website.

The subject invention also contemplates those polynucleotide molecules(encoding an inflammatory mediator of the invention) having sequenceswhich are sufficiently homologous with the polynucleotide sequencesencoding an inflammatory mediator of the invention so as to permithybridization with that sequence under standard stringent conditions andstandard methods (Maniatis, T. et al., 1982). As used herein,“stringent” conditions for hybridization refers to conditions whereinhybridization is typically carried out overnight at 20-25 C below themelting temperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt'ssolution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature isdescribed by the following formula (Beltz, G. A. et al., 1983):

Tm=81.5 C+16.6 Log [Na+]+0.41 (% G+C)−0.61 (% formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at Tm-20 C for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

As used herein, the terms “nucleic acid” and “polynucleotide sequence”refer to a deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited, wouldencompass known analogs of natural nucleotides that can function in asimilar manner as naturally-occurring nucleotides. The polynucleotidesequences include both the DNA strand sequence that is transcribed intoRNA and the RNA sequence that is translated into protein. Thepolynucleotide sequences include both full-length sequences as well asshorter sequences derived from the full-length sequences. It isunderstood that a particular polynucleotide sequence includes thedegenerate codons of the native sequence or sequences which may beintroduced to provide codon preference in a specific host cell. Thepolynucleotide sequences falling within the scope of the subjectinvention further include sequences which specifically hybridize withthe sequences coding for an inflammatory mediator of the invention. Thepolynucleotide includes both the sense and antisense strands as eitherindividual strands or in the duplex.

The subject invention also concerns kits comprising an inflammatorymediator, and/or polynucleotides encoding the inflammatory mediators, ora composition comprising an inflammatory mediator, or a compound oragent that induces production of the inflammatory mediator of theinvention in one or more containers. Kits of the invention canoptionally include pharmaceutically acceptable carriers and/or diluents.A kit of the invention can comprise one or more of fms-related tyrosinekinase 3 (Flt3) ligand, interleukin-6 (IL-6), macrophage migrationinhibitory factor (MIF), interleukin-1 (IL-1), interleukin-3 (IL-3),erythropoietin (EPO), vascular endothelial growth factor A (VEGF-A),hypoxia-inducible transcription factor (HIF-1alpha), insulin like growthfactor-1 (IGF-1), tumor necrosis factor (TNF), granulocytecolony-stimulating factor (G-CSF), granulocyte/macrophagecolony-stimulating factor (GM-CSF), macrophage colony-stimulating factor(M-CSF), and metalloproteinases. In one embodiment, a kit of theinvention includes one or more other components, adjuncts, or adjuvantsas described herein. In one embodiment, a kit of the invention includesinstructions or packaging materials that describe how to administer aninflammatory mediator or composition of the kit. Containers of the kitcan be of any suitable material, e.g., glass, plastic, metal, etc., andof any suitable size, shape, or configuration. In one embodiment, aninflammatory mediator of the invention is provided in the kit as asolid, such as a tablet, pill, or powder form. In another embodiment, aninflammatory mediator of the invention is provided in the kit as aliquid or solution. In one embodiment, the kit comprises an ampoule orsyringe containing a compound and/or agent of the invention in liquid orsolution form.

The subject invention also concerns methods for identifying a compoundthat stimulates the activities of GM-CSF and its biological activitiesin reducing cognitive impairment.

The subject invention also concerns methods for identifying a compoundthat competitively inhibits the activities of GM-CSF and its biologicalactivities in reducing cognitive impairment.

Mammalian species which benefit from the disclosed methods include, butare not limited to, primates, such as apes, chimpanzees, orangutans,humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats,guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, andferrets; domesticated farm animals such as cows, buffalo, bison, horses,donkey, swine, sheep, and goats; exotic animals typically found in zoos,such as bear, lions, tigers, panthers, elephants, hippopotamus,rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests,prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena,seals, sea lions, elephant seals, otters, porpoises, dolphins, andwhales. Other species that may benefit from the disclosed methodsinclude fish, amphibians, avians, and reptiles. As used herein, theterms “patient” and “subject” are used interchangeably and are intendedto include such human and non-human species. Likewise, in vitro methodsof the present invention can be carried out on cells of such human andnon-human species.

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description of thepresent invention and are not meant to limit the scope of the invention.

The term “neural cell” means any cell of neurological origin (e.g.,brain, spinal cord) including sensory, transmittal and motor cells fromthe central nervous system or the peripheral nervous system such as aneuron, a glial, astrocyte, etc.

The term “amyloid beta peptide” means amyloid beta peptides processedfrom the amyloid beta precursor protein (APP). The most common peptidesinclude amyloid beta peptides 1-40, 1-42, 11-40 and 11-42. Other speciesof less prevalent amyloid beta peptides are described as y-42, whereby yranges from 2-17, and 1-x whereby x ranges from 24-39 and 41.

The term “carrier” means a non-toxic material used in the formulation ofpharmaceutical compositions to provide a medium, bulk and/or useableform to a pharmaceutical composition. A carrier may comprise one or moreof such materials such as an excipient, stabilizer, or an aqueous pHbuffered solution. Examples of physiologically acceptable carriersinclude aqueous or solid buffer ingredients including phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptide;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counter ions such as sodium; and/or nonionicsurfactants such as TWEEN, polyethylene glycol (PEG), and PLURONICS.

The term “contact” or “contacting” means bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

The term “condition” or “disease” means the overt presentation ofsymptoms (i.e., illness) or the manifestation of abnormal clinicalindicators (e.g., biochemical indicators), or a genetic or environmentalrisk of or propensity for developing such symptoms or abnormal clinicalindicators.

The term “endogenous” shall mean a material that an animal naturallyproduces. Endogenous in reference to, for example and not limitation,the term “kinase” shall mean that which is naturally produced by ananimal, such as a mammal (for example, and not limitation, a human) or avirus. In contrast, the term non-endogenous in this context shall meanthat which is not naturally produced by an animal, such as a mammal (forexample, and not limitation, a human) or a virus. Both terms can beutilized to describe both “in vivo” and “in vitro” systems. For example,and not limitation, in a screening approach, the endogenous ornon-endogenous kinase may be in reference to an in vitro screeningsystem. As a further example, and not limitation, where the genome of amammal has been manipulated to include a non-endogenous constitutivelyactivated kinase, screening of a candidate compound by means of an invivo system is viable.

The term “inhibit” or “inhibiting” or “suppress” or “suppressing” or“suppressive,” in relationship to the term “response” means that aresponse is decreased or prevented in the presence of a compound asopposed to in the absence of the compound.

The term “ligand” means an endogenous, naturally occurring moleculespecific for an endogenous, naturally occurring receptor.

The term “pharmaceutically acceptable prodrugs” as used herein means theprodrugs of the compounds useful in the present invention, which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of patients with undue toxicity, irritation, allergicresponse commensurate with a reasonable benefit/risk ratio, andeffective for their intended use of the compounds of the invention. Theterm “prodrug” means a compound that is transformed in vivo to yield aneffective compound useful in the present invention or a pharmaceuticallyacceptable salt, hydrate or solvate thereof. The transformation mayoccur by various mechanisms, such as through hydrolysis in blood. Thecompounds bearing metabolically cleavable groups have the advantage thatthey may exhibit improved bioavailability as a result of enhancedsolubility and/or rate of absorption conferred upon the parent compoundby virtue of the presence of the metabolically cleavable group, thus,such compounds act as pro-drugs. A thorough discussion is provided inBundgaard (1985), Widder et al. (1985), Krogsgaard-Larsen and Bandaged(1991), Bundgard (1992), Nielsenw and Bundgaard (1988), Nakeya et al.(1984), Higuchi and Stella (1987), which are incorporated herein byreference. An example of the prodrugs is an ester prodrug. “Esterprodrug” means a compound that is convertible in vivo by metabolic means(e.g., by hydrolysis) to an inhibitor compound according to the presentinvention. For example an ester prodrug of a compound containing acarboxy group may be convertible by hydrolysis in vivo to thecorresponding carboxy group.

The term “pharmaceutically acceptable salts” refers to the non-toxic,inorganic and organic acid addition salts, and base addition salts, ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of compounds useful in thepresent invention.

The term “pharmaceutical excipients” refers to non-toxic adjuvants orcompounds which can be added to the present invention which is capableof enhancing the biologically active effects of the peptide or itsabsorbancy in the body.

The term “polypeptide” relates to a protein made up of any one of thenatural or synthetic amino acids and their equivalents. In the presentinvention, a polypeptide can mean, for example, the amino acid sequenceof a GM-CSF protein or a biologically active equivalent thereof. Incertain instances, any one of the naturally occurring amino acids can bereplaced with a functional amino acid without changing the biologicalactivity of the peptide. For example, peptides are short, sequence- andlength-specific oligomers composed of amino acids. These familiarbiomolecules are ubiquitous in living cells and assume myriad roles,including cell receptor ligand, endogenous antibiotic, and evencomponents of pulmonary surfactant. Each role assumed by a bioactivepeptide will typically correspond to a unique three-dimensionalstructure. In this way, nature has exquisitely refined bioactive peptidesequences and activities through evolution and, naturally, there hasbeen significant interest in exploiting these molecules aspharmaceutical lead compounds. Often second generation pharmaceuticaltherapies have focused on the creation of non-natural peptide mimics.These ‘peptidomimetics’ can be based on any oligomer that mimics peptideprimary structure through use of amide bond isosteres and/ormodification of the native peptide backbone, including chain extensionor heteroatom incorporation. Peptidomimetic oligomers are oftenprotease-resistant, and may have reduced immunogenicity and improvedbioavailability relative to peptide analogues. In addition to primarystructural mimicry, a select subset of the sequence-specificpeptidomimetic oligomers, the so-called ‘foldamers,’ exhibitswell-defined secondary structural elements such as helices, turns andsmall, sheet-like structures. When a peptide's bioactivity or itsbiological equivalent is contingent upon a precise 3-D structure, thecapacity of a biomimetic oligomer to fold can be indispensable. Examplesof simple peptidomimetics include azapeptides, oligocarbamates andoligoureas, and common foldamer examples include β-peptides, γ-peptides,oligo(phenylene ethyylenes), vinylogous sulfonopeptides andpoly-N-substituted glycines (peptoids). Therefore, peptidomimetics of aan inflammatory mediator of the present invention, such as GM-CSFpolypeptide are within the scope of the present invention.

The term “solvate” means a physical association of a compound useful inthis invention with one or more solvent molecules. This physicalassociation can include hydrogen bonding. In certain instances thesolvate will be capable of isolation, for example when one or moresolvent molecules are incorporated in the crystal lattice of thecrystalline solid. “Solvate” encompasses both solution-phase andisolable solvates. Representative solvates include hydrates, ethanolatesand methanolates.

The term “effective amount” or “therapeutically effective amount” meansthat amount of a compound or agent that will elicit the biological ormedical response of a subject that is being sought by a medical doctoror other clinician. In particular, with regard to treating a neuronaldisorder, the term “effective amount” is intended to mean that effectivedoses of medicament which can decrease cognitive impairment in asubject. The typical weight for an average mouse is approximately 0.025kg with a metabolic rate of approximately 7.2 times that of a human. Thetypical weight for an average person is approximately 70 kg. With thestandard weight and metabolic rate adjustments, it is within the scopeof one of ordinary skill in the art to be able to derive effective dosesfor therapies of medicament of the invention as described herein. Forexample, effective amounts within the scope of the invention areequivalent mouse doses which is within about 5 mcg/day for a period asneeded to achieve cognitive effects which is within about 2 mg/day forhumans. Alternatively, effective doses for humans can also be within therange of about 50 mcg/day to about 2 mg/day, or alternatively 50mcg/day, or 100 mcg/day, or 250 mcg/day, or 500 mcg/day, or 750 mcg/dayor 1 mg/day or 1.25 mg/day, or 1.5 mg/day or 2 mg/day, or 2.25 mg/day,or 2.5 mg/day or adjusted as needed for the weight, metabolism andmetabolic needs of the individual to at least achieve the effectivecognitive effects of such individual.

The term “treating” means an intervention performed with the intentionof reversing or preventing the development or altering the pathology of,and thereby alleviating a disorder, disease or condition, including oneor more symptoms of such disorder, disease, or condition. Preventingrefers to prophylactic or preventative measures. The related term“treatment,” as used herein, refers to the act of treating a disorder,symptom, disease or condition, as the term “treating” is defined above.

Flt3 ligand is a ligand for the FLT3 tyrosine kinase receptor andbelongs to a small group of growth factors that regulate proliferationof early hematopoietic cells. Multiple isoforms of Flt3 ligand have beenidentified. The predominant form is the transmembrane form, which isbiologically active on the cell surface. When proteolytically cleavedthe transmembrane isoform generates a soluble form, which is alsobiologically active. Flt3 ligand binds to cells expressing the tyrosinekinase receptor Flt3. Flt3 ligand alone cannot stimulate proliferation,but synergizes well with other Colony Stimulating Factors (CSFs) andinterleukins to induce growth and differentiation.

Interleukin 6 (IL-6) is a multifunctional 24 kDa protein originallydiscovered in the medium of RNA stimulated fibroblastoid cells. It isupregulated by IL-1, TNF, PDGF, IFN-beta, TNF-alpha, NGF, IL-17 anddownregulated by glucocorticoids IL-4, TGF-beta. IL-6 appears to bedirectly involved in the responses that occur after infection andcellular injury, and it may prove to be as important as IL-1 andTNF-alpha in regulating the acute phase response. IL-6 has also beenimplicated in regulating adipose mass. IL-6 is reported to be producedby fibroblasts, activated T cells, activated monocytes or macrophagesand endothelial cells. It acts upon a variety of cells includingfibroblasts, myeloid progenitor cells, T cells, B cells and hepatocytes.In addition, IL-6 appears to interact with IL-2 in the proliferation ofT lymphocytes. IL-6 potentiates the proliferative effect of IL-3 onmultipotential hematopoietic progenitors.

Macrophage migration inhibitory factor (MIF), originally described as aT lymphocyte-derived factor that inhibited the random migration ofmacrophages, is an enigmatic cytokine for almost 3 decades. In recentyears, the discovery of MIF as a product of the anterior pituitary glandand the cloning and expression of bioactive, recombinant MIF proteinhave led to the definition of its critical biological role in vivo. MIFhas the unique property of being released from macrophages and Tlymphocytes that have been stimulated by glucocorticoids. Once released,MIF overcomes the inhibitory effects of glucocorticoids on TNF alpha,IL-1 beta, IL-6, and IL-8 production by LPS-stimulated monocytes invitro and suppresses the protective effects of steroids against lethalendotoxemia in vivo. MIF also antagonizes glucocorticoid inhibition ofT-cell proliferation in vitro by restoring IL-2 and IFN-gammaproduction. This observation has resulted in the identification of apivotal role for MIF within the immune system and fills an important gapin our understanding of the control of inflammatory and immuneresponses. Glucocorticoids have long been considered to be an integralcomponent of the stress response to infection or tissue invasion andserve to modulate inflammatory and immune responses. MIF is the firstmediator to be identified that can counter-regulate the inhibitoryeffects of glucocorticoids and thus plays a critical role in the hostcontrol of inflammation and immunity.

Tumor necrosis factor (TNF) is a member of a superfamily of proteins,each with 157 amino acids, which induce necrosis (death) of tumor cellsand possess a wide range of proinflammatory actions. Tumor necrosisfactor is a multifunctional cytokine with effects on lipid metabolism,coagulation, insulin resistance, and the function of endothelial cellslining blood vessels.

Interleukin-1 (IL-1) is one of the first cytokines ever described. Itsinitial discovery was as a factor that could induce fever, controllymphocytes, increase the number of bone marrow cells and causedegeneration of bone joints. At this time, IL-1 was known under severalother names including endogenous pyrogen, lymphocyte activating factor,haemopoetin-1 and mononuclear cell factor, amongst others. It was around1984-1985 when scientists confirmed that IL-1 was actually composed oftwo distinct proteins, now called IL-1α and IL-1β.

Interleukin-3 (IL-3), a 152 amino acid protein, usually glycosylated,was originally found as a T lymphocyte-derived factor which induced20alpha-hydroxysteroid dehydrogenase synthesis within hematopoieticcells. It is secreted by activated T cells and binds to the IL-3receptor that heterodimerizes with a common beta c receptor subunit,which is shared by GM-CSF and IL-5. IL-3 functions similarly to GM-CSFand the human IL-3 gene is located on chromosome 5, nine kilobases awayfrom the GM-CSF gene. IL-3 has been shown to support the proliferationof many hematopoietic cell types and is involved in a variety of cellactivities such as cell growth, differentiation and apoptosis.

Erythropoietin (EPO) is a 30.4 kDA glycoprotein hormone, produced mainlyby peritubular fibroblasts of the renal cortex, and it works to protecterythrocytes from apoptosis, as well as promote proliferation andmaturation of erythroid progenitor cells through synergistic actionswith other growth factors (IGF-1, IL-3, and GM-CSF). Blood oxygenationis thought to regulate EPO's expression throughconstitutively-synthesized transcription factors calledhypoxia-inducible factors. EPO has also been reported to haveneuroprotective and angiogenic properties.

Vascular endothelial growth factor A (VEGF-A) is a multi-functionalprotein that belongs to the platelet-derived growth factor family and isinvolved in vasculogenesis and angiogenesis. It is produced in hypoxiccells and released by HIF-1α expression to bind to cell surfacereceptors VEGFR-1 (Flt-1) or VEGFR-2 (KDR/Flk-1), inducing variousfunctions accordingly. It has been shown to stimulate endothelial cellmitogenesis and migration, vasodilation, and be chemotactic tomonocytic-lineage hematopoietic cells.

Hypoxia-inducible transcription factor alpha (HIF-1α) is a transcriptionfactor that responds to changes in available cellular oxygen. Innormoxic conditions, it is rapidly degraded by the proteasome uponprolyl hydroxylation by HIF prolyl-hydroxylase, which subsequentlyinduces its ubiquitination. Hypoxic conditions prevents this prolylhydroxylation, and HIF-1α is stabilized and acts to upregulate severalgenes which promote hypoxic survival, such as EPO, and VEGF. HIF-1α thusincreases vasculogenesis and angiogenesis through upregulation of thesegenes.

Insulin-like growth factor 1 (IGF-1) is a 70 amino acid polypeptideprotein hormone similar in molecular structure to insulin and isproduced primarily by the liver. IGF-1 binds to the IGF-1 receptor(IGF-R) and the insulin receptor (IR), and is a potent activator of thecell growth and anti-apoptotic AKT signaling pathway. Although producedthroughout life, its expression is lowest in infancy and old age, andIGF-R and IR have been reported to be reduced in Alzheimer's disease(AD) and correlate with neurodegeneration. IGF-1 has also beenimplicated in the pathogenesis of type 2 diabetes, a positive-riskfactor for AD, through vascular complications resulting from itsdeficient signaling with insulin and EPO.

Granulocyte colony-stimulating factor (G-CSF or GCSF) is acolony-stimulating factor hormone. It is a glycoprotein, growth factoror cytokine produced by a number of different tissues to stimulate thebone marrow to produce granulocytes and stem cells. G-CSF thenstimulates the bone marrow to release them into the blood. It alsostimulates the survival, proliferation, differentiation, and function ofneutrophil precursors and mature neutrophils. G-CSF is also known ascolony-stimulating factor 3 (CSF-3).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a proteinsecreted by macrophages, T cells, mast cells, endothelial cells andfibroblasts. GM-CSF is a cytokine that functions as a white blood cellgrowth factor. GM-CSF stimulates stem cells to produce granulocytes(neutrophils, eosinophils, and basophils), monocytes, and dendriticcells. Monocytes exit the circulation and migrate into tissue, whereuponthey mature into macrophages. It is thus part of the immune/inflammatorycascade, by which activation of a small number of macrophages canrapidly lead to an increase in their numbers, a process crucial forfighting infection. The active form of the protein is foundextracellularly as a homodimer.

Macrophage colony-stimulating factor (M-CSF or CSF-1) is a secretedcytokine which influences hemopoietic monocytic cells to proliferate anddifferentiate into macrophages or other related cell types, such asosteoclasts, as well as activating mature macrophages, osteoclasts, andmicroglia. M-CSF has been recently reported to have decreased expressionin AD patients, and its peripheral administration in an amyloidosismurine animal model of AD ameliorated amyloid deposition and cognitivefunction.

Metalloproteinases (or metalloproteases) constitute a family of enzymesfrom the group of proteinases, classified by the nature of the mostprominent functional group in their active site. There are two subgroupsof metalloproteinases: exopeptidases called metalloexopeptidases andendopeptidases called metalloendopeptidases. Well knownmetalloendopeptidases include ADAM proteins and matrixmetalloproteinases.

Darbepoetin is a novel erythropoetin-stimulating factor (NESP) that hasa longer plasma half-life than EPO, and only varies by native human EPOat 5 amino acid positions. Darbepoetin would also be expected to actsynergistically with GM-CSF in the maturation and proliferation of theburst-forming and colony-forming erythroid units to the normoblast stageof erythropoiesis (Fisher (2003)). Erythropoiesis is important inAlzheimer's disease due to oxygenation of neuronal cells, and toclearance of complement-bound circulating amyloid beta proteins via theexpression of complement receptor 1 (CR1) on the erythrocyte surface(Rogers et al. (2006); Cadman and Puttfarcken (1997); Helmy et al.(2006)).

Stem Cell Factor (SCF) is a cytokine which has been reported tostimulates the growth and development of primitive multipotential andunipotential hematopoietic stem cells either alone or in combinationwith other cytokines such as GM-CSF, G-CSF, and EPO (Fisher (2003)). SCFalso acts to stimulate the function of mature granulocytes (Czygier etal. (2007)).

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1

Radial Arm Water Maze Testing—Working (short-term) memory is evaluatedin the radial arm water maze (RAWM) task, using the same pool that wasinvolved in both Morris water maze and platform recognition testing.This task also uses the same clear platform and visual cues as in Morrismaze testing.

For the radial arm water maze task of spatial working memory, analuminum insert was placed into a 100 cm circular pool to create 6radially distributed swim arms emanating from a central circular swimarea. An assortment of 2-D and 3-D visual cues surrounded the pool. Thenumber of errors prior to locating which one of the 6 swim armscontained a submerged escape platform (9 cm diameter) was determined for5 trials/day over 8 days of pre-treatment testing and 4 days ofpost-treatment testing. There was a 30-min time delay between the 4thtrial (T4; final acquisition trial) and 5th trial (T5; memory retentiontrial). The platform location was changed daily to a different arm, withdifferent start arms for each of the 5 trials semi-randomly selectedfrom the remaining 5 swim arms. During each trial (60 s maximum), themouse was returned to that trial's start arm upon swimming into anincorrect arm and the number of seconds required to locate the submergedplatform was recorded. If the mouse did not find the platform within a60-s trial, it was guided to the platform for the 30-s stay. The numbersof errors and escape latency during trials 4 and 5 are both consideredindices of working memory and are temporally similar to the standardregistration/recall testing of specific items used clinically inevaluating AD patients.

Following post-treatment completion of RAWM testing (4 days), all micewere further evaluated in a novel cognitive interference task for 6days. This task involves two radial arm water maze set-ups in twodifferent rooms, and 3 different sets of visual cues. The task requiredanimals to remember a set of visual cues, so that following interferencewith a different set of cues, the initial set of cues can be recalled tosuccessfully solve the radial arm water maze task. A set of fivebehavioral measures were examined. Behavioral measures were: A1-A3(Composite three-trial recall score from first 3 trials performed inRAWM “A”), “B” (proactive interference measure attained from a singletrial in RAWM “B”), A4 (retroactive interference measure attained duringa single trials in RAWM “A”), and “A5” (delayed-recall measure attainedfrom a single trial in RAWM “A” following a 20 minute delay between A4and A5). As with the stand RAWM task, this interference task involvedthe platform location being changed daily to a different arm for both ofthe RAWM set-ups utilized, and different start arms for each day oftesting for both RAWM set-ups. For A1 and B trials, the animal wasinitially allowed one minute to find the platform on its own before itwas guided to the platform. Then the actual trial was performed in eachcase.

Mouse recombinant GM-CSF was used in each experiment using PS/APP mice.5 μg per injection were reconstituted in saline and injected intotreated mice subcutaneously over the period of the study as outlinedbelow.

Cohort 1: 29 mice, F8 White Generation, DOB Apr. 20-May 7, 20078 APP mice, 21 Non-transgenic micePre-testing RAWM: Mice 1 year old

8 Days of RAWM: Apr. 25-May 2, 2008

15-Day delay between pre-testing and starting injections

Begin Treatment May 17, 2008

10 Days of injections (5 μg/day s.c.) before RAWM post-testing withdaily injections throughout entire testing period.Begin post-testing RAWM May 27, 2008-May 30, 2008; no testing May 31,2008-Jun. 1, 2008 but daily injections continued; begin InterferenceRAWM Jun. 2, 2008-Jun. 7, 2008Additional mice added to study=Cohort 28 mice: F8 Bright Orange, DOB May 27, 2007, 3 APP/PS1, 4 APP

F7 Light Blue, DOB Jun. 27, 2007, 1 APP Pre-testing RAWM 8 Days of RAWM:May 12-May 19, 2008

Started 17 days after cohort 115-Day delay between pre-testing and starting injections

Begin Treatment Jun. 3, 2008

10 Days of injections (5 μg/day s.c.) before RAWM post-testing withinjections each day throughout entire testing period.Begin post-testing RAWM Jun. 12, 2008-Jun. 15,2008; no testing Jun. 16,2008-Jun. 17, 2008 but daily injections continued; Begin InterferenceRAWM Jun. 18, 2008-Jun. 23, 2008

Results:

FIGS. 3, 4, and 7 depict the results of the GM-CSF testing. FIGS. 7A-7D:Standard RAWM errors (4 days of testing; two 2-day blocks). For bothBlocks 1 and 2 of testing, the untreated APP transgenic group showsclear impairment compared to all or most other groups on working memorytrials T4 and T5. Performance over all post-treatment days of testingrevealed that the untreated APP transgenic group was substantiallyimpaired compared to all 3 other groups, which performed similarly toeach other.

FIG. 7E: GM-CSF Interference Testing—Overall

The untreated APP transgenic group was very impaired in immediate recalltrials (A1-A3) compared to all 3 other groups. The untreated APPtransgenic group showed significantly impaired performance for bothproactive (Trial B) and retroactive (Trial A4) interference trialscompared to GM-CSF treated nontransgenic littermate controls. APPtransgenic GM-CSF treated group performed no differently from treatedand untreated nontransgenic littermate control groups on these trials.The untreated APP transgenic group was very impaired in the delayedrecall trial (A5) compared to all 3 other groups, which did not differfrom one another in performance.

FIGS. 3 and 4: GM-CSF Interference Testing—Block 1 and Block 2.

During both blocks, the untreated APP transgenic group showed impairedimmediate recall (A1-A3) and impaired delayed recall (A5) compared toall 3 other groups, which exhibited no differences from one another inperformance. During Block 1, the APP untreated transgenic group showed aselective impairment in proactive interference performance (Trial B).All the other groups performed much better during this trial andperformances was identical to one another. During both blocks, theuntreated APP transgenic group was impaired in retroactive interference(A4) compared to the GM-CSF treated nontransgenic littermate group.

It is evident from both standard RAWM testing and RAWM interferencetesting that the untreated APP transgenic group is consistently impairedin working memory, proactive interference, retroactive interference, anddelayed recall compared to the other 3 groups. By contrast, the GM-CSFtreated APP transgenic group's performance is never statisticallydifferent from that of the nontransgenic littermates which were GM-CSFtreated or untreated. It is clearly indicated that the untreated APPtransgenic group performed much poorer across multiple cognitivemeasures in comparison to the other 3 groups.

Further, in a separate set of experiments wherein groups of mice weretreated with caffeine and plasma levels of GM-CSF were ascertained todetermine if there was a correlation between GM-CSF levels and amyloidbeta levels in the hippocampus, it was found that there were stronginverse correlations between plasma GM-CSF and hippocampal insolubleA-beta levels, e.g. see FIGS. 5A and 5B. By contrast, there was asignificant positive correlation between plasma GM-CSF and hippocampalsoluble A-beta levels (see FIG. 5C). This separate observational studyusing animals in a different set of experiments suggested that GM-CSFwas in some way removing insoluble deposited A-beta from the brain,resulting in increased soluble A-beta in the brain. Perhaps the elevatedsoluble A-beta is the transport mechanism for clearance of the betaplaques into the plasma. This may suggest a mechanism of cognitivebenefits of GM-CSF. Other mechanisms which may explain the cognitivebenefits of GM-CSF or its biological equivalents are possible and withinthe scope of the invention as described herein, would includeneovascularization in the brain with increased cerebral blood flow,reductions in plaques and their associated inflammation,bone-marrow-derived neurogenesis, or neuroprotection againstmicrovascular plaque-induced ischemia and resulting oxidative stress. Itis also considerable that any combination of these mechanisms mentionedherein is considered as attributable to the effects of GM-CSF and itsbiological equivalents on cognitive benefits and would be within thescope of practice of the claimed invention as described herein.

Materials and Methods for Example 2

Transgenic Mice Involving Intracerebral Administration of CSFs.

PS/APP mice were generated by crossing heterozygous PDGF-hAPP (V717F)mice with PDGF-hPS1 (M146L) on both Swiss Webster and C57BL/6backgrounds. Transgene detection was performed using comparativereal-time PCR (Bio-Rad iCycler-Hercules, Calif.). The pathogenicphenotype in this model is robust amyloid plaque accumulation beginning6-8 months of age. All procedures involving experimentation on animalswere performed in accordance with the guidelines set forth by theUniversity of South Florida Animal Care and Use Committee.

Intracranial Infusions of M-CSF.

Animals (PS/APP, all 8.8-9.6 months, 25-35 g, both genders) wereanesthetized with 1-2% isoflurane, shaved and scrubbed with 10% Betadinesolution at the site of incision, and placed into a stereotaxic frame(Kopf Instruments, Tujunga, Calif.). A small (4 cm) incision was made,exposing the skull, and double bladed scissors were used to form asubcutaneous pocket along the animal's back into which osmotic minipumps(Alzet model 1004, Durect Corp., Cupertino, Calif.) were inserted. Twoholes were drilled into the skull (from Bregma −0.1 mmanterior-posterior, +/−0.9 mm medial-lateral, and the 30 gauge catheterswere inserted at depth of 3.0 mm, corresponding to the lateralventricles). Leading from the Alzet pump is a proprietary cathetersystem (International Application No. PCT/US08/73974) with the deliverytips fashioned to the contours of the skull rather than thecommercially-available pedestal cannula. This completelysubcutaneously-contained system allows bilateral intracerebral infusionof test substances. This capability overcomes the problem of thevariance in amyloid deposition between animals (see FIG. 10), byallowing for infusion into each hemisphere, where a test substance canbe delivered ipsilaterally and vehicle contralaterally, effectivelymaking each animal its own control. Moreover, the scalp heals andreduces chronic inflammation and irritation to the mice from the openwound, seen with pedestal-mounted cannula usage. Once the cannulae areboth inserted, they are affixed to the skull using Locktite 454 adhesive(Plastics One, Roanoke, Va.) and secured with 1 cm diameter nitrile.After the adhesive cures, the scalp is sutured with 6-7 silk sutures andthe animal is given an immediate dose of ketoprofen (10 mg/kg) and againevery 6 hours as needed for another 48 hours.

M-CSF was bilaterally infused directly into the lateral ventricles (5μg/day) for 14 days using Alzet model 1004 with an average flow rate of0.12 μL/hour. The pumps and catheters were primed for 48 hours in a 37°C. water bath prior to implantation. After the 14-day period, theanimals are given an overdose (˜100 mg/kg, i.p.) of sodium pentobarbitalfollowed by transcardial perfusion with 0.9% cold saline.

Stereotaxic Injection of CSFs.

All 3 CSFs were stereotaxically-injected (5 μg/injection) into the(ipsilateral) hippocampus, with vehicle (artificial cerebrospinal fluid(aCSF)) injected contralaterally into four PS/APP mice each (all 10-12months old, 25-35 g, both genders). Two holes were drilled into theskull (from bregma −2.5 mm anterior-posterior, +/−2.5 mm medial-lateral,and the 30 gauge needle inserted to a depth of 2.5 mm). Mice wereeuthanized and 0.9% cold saline-perfused 7 days later. Recombinant mouseGM-CSF (rmGM-CSF), recombinant murine G-CSF (rmG-CSF), and recombinantmouse M-CSF (rmM-CSF) (R&D Systems) will be referred to as GM-CSF,G-CSF, and M-CSF throughout this publication.

Immunohistochemistry and Image Analysis of Intrahippocampal-InjectedMice.

Perfused brain tissues from the intrahippocampus-injected mice werefixed in 10% neutral buffered formalin for 24-36 hours and then placedthrough a sucrose gradient (10-30%) over another 72 hours. Brains werethen frozen to the peltier (Physitemp, Clifton, N.J.) of the histoslide(Leica, Heerbruug, Switzerland) and sectioned coronally at 14 μm.Alternatively, selected brains were paraffin-embedded after 10% neutralbuffered formalin fixation, sectioned at 5 μm, and adhered to slides.Deparaffination and antigen retrieval (boiled in 10 mM Sodium Citratebuffer for 20 min) were performed before immunohistochemical staining Tosignificantly reduce cost of reagents and antibodies withparaffin-embedded slides, a novel magnetic immunohistochemical stainingdevice was developed. Standard immunohistochemical techniques usedanti-Aβ antibodies, i.e. 6E10 (1:1000), and MabTech's (1:5000) toimmunolabel amyloid deposition followed by Alexa 488 and/or 564secondary fluorophores (1:1000, 1:4000—Invitrogen), and Hoechst (Sigma)nuclear staining. The tissues were visualized on a Zeiss Imager.Z1fluorescence microscope with a Zeiss Axiocam Mrm camera (Oberkochen,Germany) using Axiovision 4.7 software. Photomicrographs were taken at10× and montaged with Axiovision Panarama Module to select equal areasfrom corresponding loci of each brain hemisphere. There were 5 coronalsections/mouse with 15-25 10× pictures/hemisphere analyzed (variedaccording to anterior-posterior location). Amyloid quantification wasperformed using ImageJ software program (developed at and available fromNational Institutes of Health). Briefly, each analyzed picture percoronal section was thresholded equally to the same standard deviationfrom the histogram mean, and we used the same area threshold to minimizebackground artifacts, and analyzed for area, perimeter, feret diameter,and integrated density parameters. The feret diameter is the longestdistance across a given plaque, and the integrated density is theproduct of the area and the average gray value of the plaque's pixels.

Behavioral Transgenic Mouse Study Involving GM-CSF Treatment.

Mice in this study were derived from the Florida Alzheimer's DiseaseResearch Center transgenic mouse colony, wherein heterozygous micecarrying the mutant APPK670N, M671L gene (APPsw) are routinely crossedwith heterozygous PS1 (Tg line 6.2) mice to obtain offspring consistingof APPsw/PS1, APPsw, PS1, and non-transgenic (NT) genotypes on a mixedC57/B6/SW/SJL background. Eleven APPsw, 4 APPsw/PS1, and 17 NT mice, all12-months old, were selected for use in this study, and evaluated in theRAWM task of working memory for 8 days (see protocol below). Numerousexperiments have revealed that various genotypes of AD perform equallyonce they reach cognitive impairment. Thus, the 15 Tg mice were dividedinto two groups, balanced in RAWM performance, with 2 APPsw miceincluded in each group. The 17 NT mice were also divided into twogroups, balanced in RAWM performance. Two weeks following completion ofpre-treatment testing, one group of Tg mice (n=7) and one group of NTmice (n=9) were started on a 10-day treatment protocol with GM-CSF (5μg/day given subcutaneously), while animals in the control Tg and NTgroups (n=8 per group) received daily vehicle (saline) treatmentsubcutaneously over the same 10-day period. On the 11^(th) day ofinjections, all mice began evaluation for four days in the RAWM task,given 2 days of rest, then followed by an additional 4 days of testingin the novel Cognitive Interference task (see protocol below). DailyGM-CSF and saline injections were continued throughout the behavioraltesting period. After completion of behavioral testing at 3 weeks intotreatment, all mice were euthanatized, brains fixed as described above,and paraffin-embedded. Careful visual examination of all tissues uponnecropsy revealed no morphological abnormalities, and the mice tolerateddaily subcutaneous injections well.

Immunohistochemistry and Image Analysis of Subcutaneous GM-CSF-TreatedMice.

Perfused brain tissues from the subcutaneously injected,behaviorally-tested mice were fixed in 10% neutral buffered formalin for24-36 hours, and then paraffin-embedded. At the level of the hippocampus(bregma −2.92 mm to −3.64 mm), five 5-μm sections (150 μm apart) weremade from each mouse brain using a sliding microtome, and mounted perslide. Immunohistochemical staining was performed following themanufacturer's protocol using a Vectastain ABC Elite kit (VectorLaboratories, Burlingame, Calif.) coupled with the diaminobenzidinereaction, except that the biothinylated secondary antibody step wasomitted for Aβ immunohistochemical staining. The following primaryantibodies were used for immunohistochemical staining: a biothinylatedhuman Aβ monoclonal antibody (clone 4G8; 1:200, Covance ResearchProducts, Emeryville, Calif.) and rabbit synaptophysin polyclonalantibody (undiluted, DAKO, Carpinteria, Calif.). For Aβimmunohistochemical staining, brain sections were treated with 70%formic acid prior to the pre-blocking step. Phosphate-buffered saline(0.1 mM, pH 7.4) or normal rabbit serum (isotype control) was usedinstead of primary antibody or ABC reagent as a negative control.Quantitative image analysis was done based on previous methods(Sanchez-Ramos et al. (2009)). Images were acquired using an OlympusBX60 microscope with an attached digital camera system (DP-70, Olympus,Tokyo, Japan), and the digital image was routed into a Windows PC forquantitative analysis using SimplePCI software (Compix Inc., ImagingSystems, Cranberry Township, Pa.). Images of five 5-μm sections (150 μmapart) through both anatomic regions of interest (hippocampus andentorhinal cortex) were captured from each animal, and a thresholdoptical density was obtained that discriminated staining frombackground. Each region of interest was manually edited to eliminateartifacts. For Aβ burden analysis, data are reported as percentage ofimmunolabeled area captured (positive pixels) relative to the full areacaptured (total pixels). To evaluate synaptophysin immunoreactivity,after the mode of all images was converted to gray scale, the averageintensity of positive signals from each image was quantified in the CA1and CA3 regions of hippocampus as a relative number from zero (white) to255 (black). Each analysis was done by a single examiner blinded tosample identities (T.M.).

Behavioral Tasks.

Each analysis was done by a single examiner (N.G.) blinded to sampleidentities, and statistical analyses were performed by a single examiner(M.R.) blinded to treatment group identities. The code was not brokenuntil analyses were completed.

Radial Arm Water Maze.

For the RAWM task of spatial working memory (Arendash et al. (2001);Ethell et al. (2006); Arendash et al. (2007)), an aluminum insert wasplaced into a 100 cm circular pool to create 6 radially-distributed swimarms emanating from a central circular swim area. An assortment of 2-Dand 3-D visual cues surrounded the pool. The number of errors prior tolocating which one of the 6 swim arms contained a submerged escapeplatform (9 cm diameter) was determined for 5 trials/day. There was a30-min time delay between the 4th trial (T4; final acquisition trial)and 5th trial (T5; memory retention trial). The platform location waschanged daily to a different arm, with different start arms for each ofthe 5 trials semi-randomly selected from the remaining 5 swim arms.During each trial (60 s maximum), the mouse was returned to that trial'sstart arm upon swimming into an incorrect arm and the latency timerequired to locate the submerged platform was recorded. If the mouse didnot find the platform within a 60-s trial, it was guided to the platformfor a 30-s stay. The numbers of errors and escape latency during trials4 and 5 are both considered indices of working memory and are temporallysimilar to standard registration/recall testing of specific items usedclinically in evaluating AD patients.

Cognitive Interference Task.

This task was designed to mimic, measure-for-measure, a cognitiveinterference task recently utilized clinically to discriminate betweennormal aged, MCI, and AD patients (Loewenstein et al. (2004)). The taskinvolves two RAWM set-ups in two different rooms, with two sets ofvisual cues different from those utilized in standard RAWM testing. Thetask requires animals to remember a set of visual cues (in RAWM-A), sothat following interference with a different set of cues (in RAWM-B),the initial set of cues can be recalled to successfully solve the RAWMtask. Five behavioral measures were examined: A1-A3 (Compositethree-trial recall score from first 3 trials performed in RAWM-A), B(proactive interference measure attained from a single trial in RAWM-B),A4 (retroactive interference measure attained during a single trial inRAWM-A), and A5 (delayed-recall measure attained from a single trial inRAWM-A following a 20-min delay between A4 and A5). As with the standardRAWM task, this interference task involves the platform location beingchanged daily to a different arm for both RAWM set-ups. For A1 and Btrials, the animal is initially allowed one minute to find the platformon their own before being guided to the platform. Then the actual trialis performed in each case. As with the standard RAWM task, animals weregiven 60s to find the escape platform per trial, with the number oferrors and escape latency recorded per trial.

Statistical Analysis.

Statistical analysis of amyloid plaque parameters from ipsilateralGM-CSF administration versus contralateral aCSF-injection hemisphereswas performed using paired Student t-test with a P-Value of <0.05considered significant. For statistical analysis of RAWM data, the8-days of pre-treatment testing (four 2-day blocks) or the 4-days ofpost-treatment testing (two 2-day blocks) were evaluated for individualblocks, as well as over all blocks, using one-way ANOVAs. Thereafter,post hoc pair-by-pair differences between groups were resolved with theFisher LSD (least significant difference) test. For statistical analysisof cognitive interference data, both 2-day blocks were analyzedseparately, as were all four days collectively. One way ANOVA's wereemployed for each of the four behavioral measures analyzed, followed bypost hoc Fisher's LSD test to determine significant group differences atP<0.05. Statistical analysis of amyloid plaque deposition andsynaptophysin staining from subcutaneous GM-CSF administration wasperformed using two-tailed homoscedastic Student t-test with a P valueof <0.05 considered significant.

Example 2

Alzheimer's disease is an age-related, progressive neurodegenerativedisorder that presents as increasing decline in cognitive and executivefunction. Alzheimer dementia is associated with cerebrovasculardysfunction (Humpel and Marksteiner (2005)), extracellular accumulationof amyloid β (Aβ) peptides in the brain parenchyma and vasculature walls(Rhodin et al. (2000); Scheuner et al. (1996)) (predominantly Aβ₁₋₄₂ andAβ₁₋₄₀), and intraneuronal accumulation of neurofibrillary tanglesconsisting of hyperphosphorylated Tau proteins (Rapoport et al. (2002)).Associated neuroinflammation may contribute to AD pathogenesis (Griffinet al. (1989); Akiyama et al. (2000); Wyss-Coray (2006)), as theinflammatory proteins apolipoprotein E (apoE) and α1-Antichymotrypsin(ACT) catalyze the polymerization of Aβ peptides into amyloid filamentsin vivo and in vitro (Wisniewski et al. (1994); Ma et al. (1996); Potteret al. (2001); Nilsson et al. (2004); Padmanabhan et al. (2006)).However, NSAIDs have failed to reverse or prevent AD pathology, anddystrophic microglia are suggested to precede neurodegenerative dementia(Streit et al. (2009)). It has also been shown that amyloid plaques formrapidly and then become decorated by microglia (Koenigsknecht-Talboo etal. (2008); Meyer-Luehmann et al. (2008)), both resident and bonemarrow-derived, suggesting an ability and intention to remove amyloid(Malm et al. (2005); Simard and Rivest (2004); Simard et al. (2006)).

Rheumatoid arthritis is an autoimmune disease in which inflamed synovialtissue and highly vascularized pannus forms, irreparably damaging thecartilage and bone. In this inflammatory pannus, leukocyte populationsare greatly expanded, and many proinflammatory factors are produced thatwork together in feed-forward mechanisms, further increasingleukocytosis, cytokine/chemokine release, osteoclastogenesis,angiogenesis, and autoantibody production (rheumatoid factors andanti-citrullinated protein antibodies) (Szekanecz and Koch (2007); vander Voort et al. (2005); Schellekens et al. (2000)). Additionally, theadaptive immune system presents a Th17 phenotype within CD4⁺lymphocytes, with ultimate production of interleukin (IL-17) which isthen responsible for inducing much of the pro-inflammatory effects(Parsonage et al. (2008); Cox et al. (2008)). Further enhancements ofleukocyte populations come from local expression of colony-stimulatingfactors: M-CSF (macrophage), G-CSF (granulocyte), and GM-CSF(granulocyte-macrophage) (Seitz et al. (1994); Leizer et al. (1990);Nakamura et al. (2000)).

Although up-regulated leukocytes in RA could potentially enter into thebrain and inhibit development of AD pathology and/or neuronaldysfunction, lymphocytic infiltrates into AD patient brains have notbeen reported. The lack of infiltration suggests that activation of theinnate immune system might be responsible for preventing AD pathology inRA patients. For instance, complement proteins are up-regulated in ADbrain, and inhibition of C3 convertase significantly increases amyloidpathology in AD mice (Wyss-Coray et al. (2002)). Bone marrow-derivedmicroglia play a critical role in restricting amyloid deposition, butthis association declines with age, while AD pathology increases (Simardet al. (2006)). El Khoury and colleagues have shown that microglia areactually protective against AD pathogenesis in multiple ways, i.e.,through delay of amyloidosis via chemokine receptor (CCR2) recruitment,by up-regulation of its ligand, monocyte chemotactic protein-1(MCP-1/CCL2), by induced expression of Aβ-binding scavenger receptors(CD36, scavenger receptor A, and receptor for advanced glycation endproducts), and by induced expression of Aβ-degrading enzymes[neprilysin, insulysin, and matrix metalloproteinase (MMP9)]. However,the expression of these receptors and enzymes also decreases with age(Hickman et al. (2008); El Khoury et al. (2007)).

To investigate the interplay of the innate immune system and AD, westudied the effects on AD pathology of the three structurally-unrelatedcolony-stimulating factors (M-CSF, G-CSF, and GM-CSF), which are allup-regulated in RA (Seitz et al. (1994); Leizer et al. (1990); Nakamuraet al. (2000)). These CSFs enhance the survival of their respectiveleukocytes and drive their proliferation and differentiation frommonocytic precursors. M-CSF and G-CSF induce specific subsets of theinnate immune system, while GM-CSF induces the full range of innatecells. Using bilateral intracerebroventricular infusion of M-CSF for twoweeks into PS/APP mice, we first examined M-CSF's effect on plaquedeposition. Immunohistochemical analysis showed considerable variancesof amyloid deposition between mice of similar age (FIG. 10),significantly compromising our ability to determine M-CSF's effect in alimited mouse cohort. While improving our drug delivery system bydeveloping novel bilateral brain infusion catheters, we found thatparenchymally-infused recombinant peptides remained localized to theinfused hemisphere. These findings led us to administer the CSFs as aunilateral intrahippocampal bolus with a contralateral injection ofvehicle as control, thus obviating the need for large numbers oftransgenic mice and age-matched littermate controls to obtainstatistical significance. Each CSF was stereotaxically injected into thehippocampus of 4 mice, with artificial cerebrospinal fluid vehicle(aCSF) injected contralaterally. The mice were sacrificed 7 dayspost-injection.

Remarkably, M-CSF injections resulted in visible swelling of the entiretreated hemisphere, noticeable fragility on sectioning, and in onemouse, an apparent hyperplasia at the injection site (FIG. 11C).Overexpression of M-CSF and/or its receptor in mammary glands hassimilarly resulted in tumor formation and hyperplasia (Kirma et al.(2004)). Amyloid plaque loads were not significantly changed in theM-CSF-injected hemispheres as compared to the control sides (data notshown). However, Boissonneault et al. (2009) published that chronicintraperitoneal (i.p.) injection of M-CSF prevents and reverses amyloiddeposition and cognitive impairment. Using GFP-expressing bone marrow,the authors also found that M-CSF induced a significant accumulation ofbone marrow-derived microglia (Boissonneault et al. (2009)). Differencesbetween these data and ours point to different study lengths and dosageeffects, with Boissonneault et al. delivering chronic i.p. 1.3 μg M-CSFper injection, compared to our 5 μg intrahippocampal bolus.

In contrast to M-CSF, G-CSF injections did not induce swelling andshowed some modest reductions of amyloid deposition (FIGS. 12A and 12B),which was corroborated by independent observations by fellowinvestigators (Sanchez-Ramos et al. (2009)). However, GM-CSF injectionsresulted in pronounced amyloid reductions, as compared to controlhemispheres (FIGS. 13A-13D), and therefore all our subsequentexperiments focused on studying the effects of GM-CSF in AD mice.Quantification of amyloid plaques revealed significant reductions withinindividual mice and overall significant reductions for all plaqueparameters measured (FIG. 6B and FIGS. 14A-14D-1). The feret diameterand the integrated density parameters were reduced throughout theGM-CSF-injected hemispheres of all 4 mice, indicating that the overallplaque sizes and dense cores were reduced simultaneously. The percentreduction in plaque deposition between individual sections of the samemouse and between different mice varied within a range of 8 to 62% (datanot shown) and thereby further highlights the variance of amyloiddepositions throughout each brain and between multiple mice.

Based upon these pathology data, we investigated the effect ofsubcutaneous GM-CSF injection on AD pathology and cognitive function.Prior to GM-CSF treatment, APPsw+PS1(Tg) mice were first confirmed byRAWM testing to be cognitively-impaired for working memory. Both thenon-transgenic control mice (NT) and the Tg mice were then sub-dividedinto two cognitively-balanced groups, for either GM-CSF or salinetreatment. RAWM testing post-injection re-confirmed that Tg control micewere substantially impaired compared to NT control mice. This impairmentwas evident in individual blocks of testing, but also over all 4 days oftesting (FIGS. 7A-7D). In sharp contrast, GM-CSF-treated Tg miceperformed equally well or better than NT control mice during individualblocks and overall. GM-CSF-treated NT mice performed as well as orslightly better than NT controls (FIGS. 7A-7D).

Before evaluation in the Cognitive Interference Task, the mice restedtwo days. This task mimics human interference testing, whichdistinguishes mild cognitive impairment (MCI) patients from agedcontrols with a high degree of accuracy (Loewenstein et al. (2004)). Inall four cognitive interference measures assessed over 4 days of testing(FIG. 7E), Tg control mice were clearly impaired compared to NT mice,and Tg mice treated with GM-CSF exhibited significantly better 3-trialrecall and delayed recall compared to Tg controls. Indeed, for all fourcognitive measures, GM-CSF-treated transgenic AD mice performedsimilarly to NT mice. A particularly strong effect of GM-CSF treatmentin Tg mice was evident for the proactive interference measure during thefirst half of testing (FIG. 7F), wherein GM-CSF-treated Tg miceperformed substantially better than Tg controls and identically to bothgroups of NT mice. Susceptibility to proactive interference has beenreported to be a more sensitive marker for differentiating MCI and ADpatients from aged normals than traditional measures of delayed recalland rate of forgetting (Loewenstein et al. (2004)). Parenthetically,even the GM-CSF-treated NT mice showed a trend towards improvedcognition in behavioral studies, albeit not statistically significant.Subsequent analysis of brains from Tg mice of this study revealed thatGM-CSF treatment induced large reductions in amyloid burdens withinentorhinal cortex (Λ55%) and hippocampal (Λ57%) compared to control Tgmice (FIG. 8E).

The improved cognitive function and reduced cortical amyloidosis ofGM-CSF-treated Tg mice were paralleled by increased synaptophysinimmunoreactivity in both CA1 and CA3 (FIGS. 9A-9E), indicating increasedsynaptic density in these hippocampal areas. Prior work has shown thatadult neural stem cells in hippocampal dentate gyms (DG) express GM-CSFreceptors, and GM-CSF increases neuronal differentiation of these cellsin a dose-dependent fashion (Kruger et al. (2007)). Thus, one mechanismfor the observed GM-CSF-induced cognitive improvement is enhancedremoval of deposited Aβ in hippocampus, with ensuing neuronalgrowth/synaptic differentiation of DG mossy fiber innervation to CA3,resulting in increased innervation/synaptogenesis of Schaffercollaterals into CA1. Removal of deposited Aβ from entorhinal cortex mayalso increase perforant pathway viability to hippocampal projectionfields in DG and CA1. Thus GM-CSF-induced enhancement ofhippocampal/entorhinal cortex circuitry, critical for working(short-term) memory, may underlie GM-CSF's reversal of working memoryimpairment in Alzheimer's Tg mice.

Example 3

Four 10-12 month old PS/APP mice were stereotaxically injected with 5 μgrmGM-CSF into the hippocampus in one hemisphere of the brain and withvehicle (aCSF: artificial cerebrospinal fluid) contralaterally. Micewere euthanized and saline-perfused 7 days later, fixed with 10%Formalin, and either cryosectioned at 14 μm or paraffin-embedded andsectioned at 5 μm. Standard immunohistochemical techniques used 6E10,and MabTech's anti-Aβ antibodies to label amyloid deposition. Microscopyand image processing were performed on a Zeiss ImagerZ1 using Axiovisionsoftware. ImageJ was utilized to quantify amyloid deposition.

Sixteen 10-12 month old PS/APP mice and sixteen non-transgenicage-matched controls were cognitively pretested by RAWM, a workingmemory paradigm. Eight mice from each group were semi-randomly chosenfor sub-cutaneous injections (5 μg/day) of rmGM-CSF. The other halfreceived daily injections of vehicle (saline). There was a 15 day restperiod after pre-testing before daily injections began. Mice wereinjected for 10 days prior to 4 days of RAWM post-testing. There was a 2day rest period before 6 days of Cognitive Interference task testing.Injections were given throughout testing and more than 1 hour beforebehavioral tests were performed. Behavioral testing and analysis wereperformed by separate staff, who were blinded to group identities.

Results:

Bolus intrahippocampal injection of rmGM-CSF reduced amyloid plaquedeposition, throughout the respective brain hemispheres, by as much as60%. In the rmGM-CSF sub-cutaneous injections, PS/APP mice, shown to becognitively impaired compared to controls, significantly reversed theircognitive impairment in the standard RAWM task. In the CognitiveInterference task, the PS/APP mice that received rmGM-CSF showed similarresults as that of the non-transgenic mice in all 4 cognitive measures.However, reductions in amyloid plaque deposition were not observed inthe sub-cutaneous study.

GM-CSF significantly reverses Alzheimer's disease-like pathology andimproves cognition in vivo.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

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1-18. (canceled)
 19. A method for reducing the level of an amyloid beta(Aβ) peptide in the brain of a person or animal comprising administeringto the person or animal an effective amount of a granulocyte/macrophagecolony-stimulating factor (GM-CSF) or a functional fragment or afunctional variant of GM-CSF that exhibits substantially the samebiological activity as GM-CSF.
 20. The method of claim 19, wherein theGM-CSF is administered intracranially.
 21. The method of claim 19,wherein the GM-CSF is administered by intracranial infusion.
 22. Themethod of claim 19, wherein the GM-CSF is administered by subcutaneous,intradermal, intravenous, intramuscular, intraperitoneal, orintrasternal injection.
 23. The method of claim 19, wherein the GM-CSFis administered to a non-neural cell or tissue.
 24. The method of claim19, wherein said method further comprises evaluating the person oranimal for cognitive impairment prior to treatment.
 25. The method ofclaim 19, wherein the GM-CSF is administered as a composition.
 26. Themethod of claim 25, wherein the composition comprises a physiologicallyor pharmaceutically acceptable carrier, diluent, or solute.
 27. Themethod of claim 19, wherein the Aβ peptide is a peptide designated as Aβpeptide 1-40, Aβ peptide 1-42, Aβ peptide 11-40, or Aβ peptide 11-42.28. The method of claim 19, wherein the GM-CSF is administeredcontinuously to the person or animal over a period of time.
 29. Themethod of claim 19, wherein the GM-CSF is administered at distinctintervals to the person or animal over a period of time.
 30. The methodof claim 19, wherein the GM-CSF is human GM-CSF.
 31. The method of claim19, wherein the Aβ peptide is present as an extracellular amyloidplaque.
 32. A method for improving cognition in a person or animalcomprising administering to the person or animal an effective amount ofa granulocyte/macrophage colony-stimulating factor (GM-CSF) or afunctional fragment or a functional variant of GM-CSF that exhibitssubstantially the same biological activity as GM-CSF, whereby the personor animal exhibits improved cognition relative to the level of cognitionexhibited by the person or animal prior to administration of the GM-CSF.33. The method of claim 32, wherein the GM-CSF is administeredintracranially.
 34. The method of claim 32, wherein the GM-CSF isadministered by intracranial infusion.
 35. The method of claim 32,wherein the GM-CSF is administered by subcutaneous, intradermal,intravenous, intramuscular, intraperitoneal, or intrasternal injection.36. The method of claim 32, wherein the GM-CSF is administered to anon-neural cell or tissue.
 37. The method of claim 32, wherein saidmethod further comprises evaluating the person or animal for cognitiveimpairment prior to treatment.
 38. The method of claim 32, wherein theimprovement in cognition is exhibited as improved learning or memorycapability.
 39. The method of claim 32, wherein the GM-CSF isadministered as a composition.
 40. The method of claim 39, wherein thecomposition comprises a physiologically or pharmaceutically acceptablecarrier, diluent, or solute.
 41. The method of claim 32, wherein theGM-CSF is administered continuously to the person or animal over aperiod of time.
 42. The method of claim 32, wherein the GM-CSF isadministered at distinct intervals to the person or animal over a periodof time.
 43. The method of claim 32, wherein the GM-CSF is human GM-CSF.